Genome editing of the susceptibility gene <scp><i>ZmNANMT</i></scp> confers multiple disease resistance without agronomic penalty in maize

Abstract

Maize is one of the most important crops in the world. However, great yield losses have been caused by several major diseases worldwide, including southern leaf blight (SLB), northern leaf blight (NLB) and Fusarium stalk rot (FSR) (Mueller et al., 2020). Identification of resistance genes and cultivation of genetic materials conferring multiple disease resistance (MDR) is one of the most effective ways to control diseases and reduce yield losses (Li et al., 2020). However, limited resistance genes conferring MDR have been identified. Recently, it has been proved that generation of genome editing materials using CRISPR/Cas9 by targeting susceptibility (S) genes (e.g. Mlo and DMR6) is a powerful way to confer disease resistance in economically important crops (Tripathi et al., 2021; Wang et al., 2014). Therefore, it is urgent to identify novel S genes. The nicotinate N-methyltransferase 1 (NANMT1) is responsible for the conversion from nicotinate to trigonelline (also known as N-methyl nicotinate) in different plant species (Li et al., 2017). We previously identified that maize ZmNANMT suppressed the hypersensitive response (HR) mediated by the auto-active nucleotide-binding, leucine-rich repeat (NLR) protein Rp1-D21 when they were transiently co-expressed in Nicotiana benthamiana (Liu et al., 2021). Maize lines carrying heterozygous Rp1-D21 exhibit autoimmune phenotypes, including growth retardation and HR lesions in the absence of pathogens (Smith et al., 2010). To further verify whether ZmNANMT can suppress Rp1-D21-mediated HR in maize, we generated ZmNANMT overexpression lines by constructing ZmNANMT into the pXG071 vector, which can distinguish the positive and negative transgenic seeds by eye observation due to the aleurone layer specific expression of DsRed2 fluorescent marker (Sun et al., 2023). From 15 independent transgenic lines, we obtained three homozygous ZmNANMT over-expression T3 lines (OE-3, OE-4 and OE-11) and their corresponding negative siblings (Neg-3, Neg-4 and Neg-11) as detected by western blot (Figure S1a). We crossed OE-3 and Neg-3 into the line containing heterozygous Rp1-D21 (Appendix S1). The resulting F1 plants showed 1:1 segregation for Rp1-D21, including OE-3 × Rp1-D21, OE-3 × no Rp1-D21, Neg-3 × Rp1-D21 and Neg-3 × no Rp1-D21. At the V6 stage grown in the field, Neg-3 × Rp1-D21 plants showed very clear HR lesions on the 6th and 7th leaves (Figure 1a,b). While in OE-3 × Rp1-D21 plants, the HR lesion phenotype was weak compared with the same leaves from their corresponding negative siblings (Figure 1a,b). At the tasselling stage, we measured the plant height and found that the over-expressing OE-3 × Rp1-D21 plants were significantly higher than their corresponding negative Neg-3 × Rp1-D21 plants (Figure 1c). These results indicated that ZmNANMT plays a negative role in the NLR protein Rp1-D21-mediated HR in maize and overexpression of ZmNANMT partially overcomes the autoimmune phenotypes conferred by Rp1-D21. To determine the role of ZmNANMT in maize disease resistance, we investigated the phenotype of the ZmNANMT over-expression lines to several maize major diseases, including SLB, NLB and FSR caused by Cochliobolus heterostrophus, Setosphaeria turcica and Fusarium verticillioides, respectively. We found that OE-3, OE-4 or OE-11 were more susceptible to SLB, NLB and FSR than their corresponding wild type siblings in the field condition (Figure 1d–f). These results indicated that ZmNANMT is a susceptibility gene in response to multiple diseases in maize. To investigate whether genome engineering of ZmNANMT can enhance MDR, we designed two single guide RNAs (sgRNA1 and sgRNA2) to target ZmNANMT (Figure S1b) using the CRISPR/Cas9 system. From 12 independent transgenic events, we obtained 3 homozygous gene editing lines; CR-6 (1 bp insertion at both sgRNA1 and sgRNA2), CR-8 (1 bp insertion at sgRNA1) and CR-10 (31 bp deletion close to sgRNA1) (Figure S1b). All the three lines were Cas9 free and with predicted open read frame shifted (Figure S1c). We investigated the responses of the T2 or T3 plants to different diseases in different conditions and found that the CR lines were significantly more resistant to FSR, SLB and NLB than their corresponding wild-type siblings, respectively (Figure 1g–i). To determine whether ZmNANMT had any negative effects on the key agronomic traits of maize, we grew the OE lines and CR lines in the field at 2 years. We found that the OE lines and the editing lines had no significant difference with their corresponding wild-type siblings for the ear size and other agronomic traits (Figure 1j,k, Figures S2 and S3). In summary, we identified that ZmNANMT is a novel plant susceptibility gene and genome editing it by CRISPR/Cas9 causes a quantitative effect on increasing MDR in maize without agronomic penalty. NANMT can convert nicotinate to trigonelline, and its function is conserved in different plant species (Li et al., 2017). We speculate that the increased MDR in CR lines might be caused by the accumulation of NA, which can increase plant disease resistance (Alnefaie et al., 2023). Genome editing of NANMT provides valuable genetic resources for increasing MDR in maize and potentially in other plant species. This research is supported by grants from the National Key Research and Development Program of China (2022YFD1201802), the National Natural Science Foundation of China (32072405 and 31871944) and the Key R&D Project of Shandong Province (2021LZGC022). The authors declare no conflict of interests. GW conceived and designed the experiments. YL, JG, SM, YX and ML performed the experiments. CZ and XL helped to analyse the data. GW wrote the manuscript. Appendix S1 Materials and methods. Figure S1 The protein detection of the overexpression lines and the sequences of three CRISPR/Cas9 lines of ZmNANMT. Figure S2 The agronomic traits of the overexpression lines of ZmNANMT. Figure S3 The agronomic traits of the CRISPR/Cas9 lines of ZmNANMT. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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